Molecular Mechanism Involved In Benzimidazole Resistance Of Beauveria Bassiana And Its Exploitation For Enhanced Resistance Of The Fungal Species

Entomopathogenic hyphomycetes are important fungal biocontrol agents against insect pests,being well represented by the fungal species Beauveria bassiana.It is well known that the field persistence of fungal formulations containing infective cells(e.g.,conidia, blastospores and/or mycelia)as active ingredients is generally affected by environmental temperature,humidity and the use of fungicides.Benzimidazole fungicides,such as benomyl,carbendazim(= bavistin,MBC)and nocodazole,have been widely used against fungal diseases of crops,yielding general resistance in phytopathogenic fungi.Thus,it is necessary to understand possible mechanisms involved in the MBC-resistance of the fungal biocontrol agents and to explore technical approaches to improving ecological fitness of fungal formulations to crop environments for better control of insect pests.This study sought to evaluate quantitatively conidial MBC-resistance of 20 Beauveria bassiana wild strains,to locate the mutated sites ofβ-tubulin gene TUB1 in MBC-resistant mutants from chemical mutagenesis of wild stain Bb2860,and to explore possible means to improving the MBC resistance of the fungal agent but not affecting its tolerance to other stressful factors. The results are summarized as follows.The increase of MBC resistance and the decrease of thermotolerance caused byβ-tubulin mutation of B.bassiana.An assay protocol to evaluate quantitatively conidial MBC-resistance was developed by counting colony forming units(CFU)on SDAY plates at gradient MBC concentrations(C).For each of 20 B.bassiana strains with different host and geographic origins,the ratio of CFU count on MBC-inclusive SDAY plates over that on MBC-exclusive ones was defined as survival index I_s.The I_s-C relationship fit well to the logistic equation I_s=1/[1+exp(α+βC)](r²â‰¥0.93).For a given isolate,the fitted equation was used to estimate median effective concentration(EC₅₀)to reduce 50%CFU count on the MBC-inclusive plates.This was a characteristic index for the conidial MBC-resistance of each isolate.Among the tested strains,19 were highly sensitive to MBC with the EC₅₀s of＜2μg/ml;only one had an EC₅₀of 2.73μg/mL,which indicates slight resistance.The strain Bb2860(EC₅₀=0.73μg/ml)was used as a parental strain for sequential chemical mutation. The MBC resistances of 11 second-run mutants based on the first-run Bb2860-M2 were enhanced by at least 758 times.The I_s observations were fitted to the logistic equation I_s= 1/[1+exp(α+βC)]for the first-run mutants(r²â‰¥0.93)but to the density equation I_s =(α-βC)-^1/λfor the second-run mutants(r²â‰¥0.92)due to their extraordinarily high resistance. All the mutants showed stable MBC resistance after three consecutive cultures on MBC-exclusive plates.The I_s observations of Bb2860 and its mutants declined with the length of time(t)after their conidia were separately exposed to 48℃were fitted to the logistic equation I_s= 1/[1+exp(α+βt)](r²â‰¥0.97).As a result,the LT₅₀s of the mutants fell in a range of 1.8-9.6 min,being much shorter than 36 min of the wild strain.The contents of hydrophobin-like proteins on conidial walls of all the mutants were also lower than that of the wild strain but little correlation was found between the contents and the LT₅₀s.However,an exponential relationship between the LT₅₀and EC₅₀was significant(LT₅₀=34.44·(EC₅₀)^-0.221,r²=0.96) among the mutant strains whose EC₅₀s were below 1000μg/ml MBC.Based on the 'BLASTP' analysis,theβ-tubulin generally present in fungi has 95% similarity.Compared to theβ-tubulin sequence of the wild strain,mutations of 1-8 amino acid residues were found in the same sequence of a given B.bassiana mutant.Of those,the three residues Q134,F167 and E198(particularly E198)are well known in relation to fungal resistance or tolerance to benzimidazole.The mutations of Q 134 are considered to be associated with heat sensitivity in phytopathogenic fungi.The mutations of other 37 amino acid residues were also found in theβ-tubulin sequences of 13 B.bassiana mutants.β-tubulin mutation versus abnormal mitosis in germinating eonidia of a seleeted mutant.To explore a relationship between MBC resistance or thermotolerance and mitosis, three assays were carried out including mitotic arrest at restrictive temperature,mitotic rescue by MBC and the fluorescence microscopy of microtubules and nuclei of the Bb2860m2-2 with high MBC resistance and low thermotolerance.The ratio of binucleated or coenocytic germinating conidia and germ tubes in all conidia was defined as chromatin / chromosome mitosis index(CMI)for evaluation.The mean 4-day colony diameter of the mutant was only 1.1 mm on MBC-exclusive SDAY plates at 30℃,but increased to 3.76 mm on SDAY plate containing 1μg/ml MBC.Under thermal stresses at 32-48℃,inclusion of MBC increased conidial germination of the mutant at 32,35 and 38℃,but did not at 400℃or higher.Based on immunofluoreseence microscopy,MBC and restrictive temperature caused absence of all microtubules in the germinating conidia of the wild strain Bb2860.For the mutant,however,abnormal microtubules were present at 25℃,and theβ-tubulin tended to be much more abundant at the tips of germ tubes but was not uniformly distributed in other parts of the tubes.In a treatment of 1μg/ml MBC at 32℃,the distribution of mutant β-tubulin was improved in the germ tubes while the uniform distribution ofα-tubulin was conspicuous.The results suggest that theβ-tubulin mutation cause abnormal changes of microtubules.The mutant microtubules could be disassembled under the influences of the restrictive temperature and MBC.Site-directed mutations for heat sensitivity and MBC resistance.To confirm the function of mutated amino acid residues inβ-tubulin,site-specific mutations of four amino acids were separately induced at Q134 and E198 using the method of splicing-by-overlap extension(SOE).A plasmid vectoring the modified TUB1 genes and the phosphinothricin resistance gene bar was constructed for use in blastospore-based transformation.As a result, the LT₅₀s of the transformants Q134K,Q134E,Q134G and Q134C were no more than 5 min at 48℃and their MBC resistance was enhanced inconspicuously.The EC₅₀of the most MBC resistant Q134G was only about twice that of the wild strain.In contrast,the transformants E 198D,E 198G,E 198K and E 198C showed significantly higher tolerance to 48℃.The LT₅₀of E198C(34.3 min)was similar to that of Bb2860(36.0 min)or the control transformant pAN52-Bar(36.3 min).The LT₅₀s of three other E198 transformants ranged from 23.3 to 29.4 min.The MBC resistances of the E198 transformants were enhanced by about 10 times compared to the wild strain.Therefore,the site-directed mutations of B. bassianaβ-tubulin indicate that mutations of the site Q134 is more associated with alteration in conidial thermotolerance whereas those of the site E198 is more associated with variation in MBC resistance.Mutation residue E198 enhanced MBC-resistance excluding heat-sensitivity.In summary,the present study has resulted in the establishment of a quantitative protocol for evaluating conidial MBC resistance of B.bassiana and the evident relationship between TUB1 and MBC resistance.Chemical mutation led to extraordinarily high MBC resistance in mutants.The site-directed mutations of Q34 and E198 provide an approach to integrating expected thermotolerance and MBC resistance into a genetically engineered transformant for improved formulation.These provide new insights into the stress biology of B.bassiana and the potential for improving field persistence of fungal formulations.